"
Team:Berkeley/Methods
From 2013.igem.org
| Line 22: | Line 22: | ||
<p> | <p> | ||
| - | <h4>Purpose:</h4 | + | <h4>Purpose:</h4> |
<p>The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: <a href="https://2013.igem.org/Team:Berkeley/Project/FMO#4" _target="new">FMO</a> and <a href="https://2013.igem.org/Team:Berkeley/Project/GLU#3" _target="new">GLU</a>) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes. </p> | <p>The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: <a href="https://2013.igem.org/Team:Berkeley/Project/FMO#4" _target="new">FMO</a> and <a href="https://2013.igem.org/Team:Berkeley/Project/GLU#3" _target="new">GLU</a>) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes. </p> | ||
<br> | <br> | ||
| - | <h4>Materials:</h4 | + | <h4>Materials:</h4> |
<ul> | <ul> | ||
<li>Purified Enzyme: FMO or GLU </li> | <li>Purified Enzyme: FMO or GLU </li> | ||
| Line 36: | Line 36: | ||
</ul> | </ul> | ||
| - | <h4>Methods:</h4 | + | <h4>Methods:</h4> |
<b>1. Determining the Concentration of Purified Enzyme</b> | <b>1. Determining the Concentration of Purified Enzyme</b> | ||
<p>Note: We followed the protocol from <a href=” http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf” _target=”new”>Bio-Rad’s Bradford Assay</a> to determine the concentration of purified FMO and GLU.</p> | <p>Note: We followed the protocol from <a href=” http://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf” _target=”new”>Bio-Rad’s Bradford Assay</a> to determine the concentration of purified FMO and GLU.</p> | ||
<p>We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.</p> | <p>We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.</p> | ||
| + | |||
| + | <b>2. Determining the Michaelis Menten kinetics </b> | ||
<div style="text-align:center"> | <div style="text-align:center"> | ||
| + | |||
</div> | </div> | ||
| + | <p> | ||
| + | Note: We assumed that the oxidation of indoxyl to indigo is much faster than that of the turnover rate of indole to indoxyl by FMO. | ||
</p> | </p> | ||
<br></div> | <br></div> | ||
Revision as of 21:29, 27 October 2013
Purpose:
The Berkeley iGEM team 2013 has determined the enzyme kinetics of FMO and GLU (data for: FMO and GLU) based on the Michaelis-Menten kinetics model. Our purpose of determining kinetics was to give a quantitative data of how fast these enzymes can turnover their respective substrates, which is very useful when considering scaling up the process and for future iGEM teams when they use our enzymes.
Materials:
- Purified Enzyme: FMO or GLU
-
- Substrate:
- FMO: Indole, NADPH (co-factor)
- GLU: Indican
- TECAN Plate Reader and TECAN Plate
- Solvents: DMSO, Water
Methods:
1. Determining the Concentration of Purified EnzymeNote: We followed the protocol from Bio-Rad’s Bradford Assay to determine the concentration of purified FMO and GLU.
We determined the concentration of our purified enzyme to be 4.2 mg/L for FMO and 3.5 mg/L for GLU.
2. Determining the Michaelis Menten kineticsNote: We assumed that the oxidation of indoxyl to indigo is much faster than that of the turnover rate of indole to indoxyl by FMO.
